Targeted molecular profiling of rare genetic alterations in colorectal cancer using next-generation sequencing

Mutation frequencies of common genetic alterations in colorectal cancer have been in the spotlight for many years. This study highlights few rare somatic mutations, which possess the attributes of a potential CRC biomarker yet are often neglected. Next-generation sequencing was performed over 112 tumor samples to detect genetic alterations in 31 rare genes in colorectal cancer. Mutations were detected in 26/31 (83.9 %) uncommon genes, which together contributed toward 149 gene mutations in 67/112 (59.8 %) colorectal cancer patients. The most frequent mutations include KDR (19.6 %), PTEN (17 %), FBXW7 (10.7 %), SMAD4 (10.7 %), VHL (8 %), KIT (8 %), MET (7.1 %), ATM (6.3 %), CTNNB1 (4.5 %) and CDKN2A (4.5 %). RB1, ERBB4 and ERBB2 mutations were persistent in 3.6 % patients. GNAS, FGFR2 and FGFR3 mutations were persistent in 1.8 % patients. Ten genes (EGFR, NOTCH1, SMARCB1, ABL1, STK11, SMO, RET, GNAQ, CSF1R and FLT3) were found mutated in 0.9 % patients. Lastly, no mutations were observed in AKT, HRAS, MAP2K1, PDGFR and JAK2. Significant associations were observed between VHL with tumor site, ERBB4 and SMARCB1 with tumor invasion, CTNNB1 with lack of lymph node involvement and CTNNB1, FGFR2 and FGFR3 with TNM stage. Significantly coinciding mutation pairs include PTEN and SMAD4, PTEN and KDR, EGFR and RET, EGFR and RB1, FBXW7 and CTNNB1, KDR and FGFR2, FLT3 and CTNNB1, RET and RB1, ATM and SMAD4, ATM and CDKN2A, ERBB4 and SMARCB1. This study elucidates few potential colorectal cancer biomarkers, specifically KDR, PTEN, FBXW7 and SMAD4, which are found mutated in more than 10 % patients.     

Targeted next-generation sequencing identified novel mutations in triple-negative myeloproliferative neoplasms

Mutations in JAK2, MPL and CALR genes have been identified in the majority of myeloproliferative neoplasm (MPN) patients, and patients negative for these three mutations are the so-called triple-negative (TN) MPN. In this study, we examined the mutational profiles of 16 triple-negative MPN patients including 7 essential thrombocythemia (ET), 1 primary myelofibrosis and 8 polycythemia vera (PV). Targeted next-generation sequencing was performed using the ACTOnco Comprehensive Cancer Panel (Ion AmpliSeq Comprehensive Cancer Panel, Life Technologies) to target all coding exons of 409 cancer-related genes. Overall, 30 nonsynonymous somatic mutations were detected in 12 (75%) patients with a range of 1–5 mutations per sample. Notably, one ET patient was found to have JAK2V617F and KITP551L mutations at very low allele frequency. One MPLP70L and 1 MPLM602T mutations were identified each in 1 ET and 1 PV, respectively. Other recurrent mutations were also identified including KMT2C, KMT2D, IRS2, SYNE1, PDE4DIP, SETD2, ATM, TNFAIP3 and CCND2. In addition, germline mutations were also found in some cancer-related genes. Copy number changes were rare in this cohort of TN MPNs. In conclusion, both somatic and germline mutations can be detected in TN MPN patients.     

晚期恶性肿瘤基因突变状态二代测序临床意义分析

目的 靶向治疗是晚期恶性肿瘤的重要治疗方法,二代测序能够准确、高通量地检测基因突变情况,对恶性肿瘤治疗有重要意义.本研究运用二代基因测序(next-generation sequencing,NGS)技术检测晚期恶性肿瘤的基因突变情况,并初步分析错义突变的临床意义.方法 2011-09-01-2016-09-30收集陕西省人民医院肿瘤内科93例晚期恶性肿瘤患者病理组织石蜡标本,利用离子个体化基因检测仪(Ion Personal Genome Machine,Ion Torrent PGM)平台检测标本16个肿瘤相关基因428个常见的突变位点的突变状态,并查询临床试验(Clinical Trails)与美国食品与药物管理局(Food and Drug Administration,FDA)官网数据资料.结果 共发现119个错义突变,其中TP53发生频率最高为34.5％(41/119);除TP53突变在各瘤种中均占较大比例外,肺癌突变频率最高为表皮生长因子受体(epidermal growth factor receptor,EGFR) 25.7％(9/35),结直肠癌为KRAS 31.6％ (6/19),胃癌为KDR 3/6,卵巢癌为KRAS 2/7,宫颈癌为KDR 3/5.70例(75.3％,70/93)检测发现＞1个的错义突变位点;93.8％(15/16)的被检测基因有正在研发中的小分子抑制剂和(或)单抗类制剂,75.0％(12/16)的被检测基因已有FDA批准用于特定瘤种的靶向药物,68.8％(11/16)的被检测基因有尚未被FDA批准的靶向药物.结论 晚期恶性肿瘤基因错义突变发生率较高,且不同瘤种的突变谱不同,目前基于NGS指导的恶性肿瘤个体化靶向治疗有广阔的应用前景.     

Targeted methylation sequencing of plasma cell-free DNA for cancer detection and classification

BackgroundTargeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis.Patients and methodsWe developed a comprehensive methylation sequencing assay targeting 9223 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we carried out a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small-cell lung cancer (NSCLC), breast cancer or melanoma and compared results with patients’ outcomes.ResultsMedian methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 versus 85.29; P = 0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 57 (83.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (78.9%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 versus 4.4 months, P < 0.001) and longer time-to-treatment failure (2.8 versus 1.6 months, P = 0.016).ConclusionsComprehensive targeted methylation sequencing of 9223 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.     

Targeting lethal minimal residual disease in small cell lung cancer

In the last three decades, treatment for small cell lung cancer has improved with advances in chemotherapy and radiotherapy. Almost all patients respond initially to standard chemotherapy, and some patients with limited stage disease are cured with the combination of chemotherapy and thoracic irradiation. Nonetheless, the majority of patients will experience lethal relapse from chemotherapy-resistant micrometastatic disease, and this has resulted in poor long-term survival for most patients. Addressing the problem of relapse requires unique approaches to eliminating minimal residual disease. This review will focus on the detection of minimal residual disease as well as strategies with which to treat it, including matrix metalloproteinase inhibitors, tyrosine kinase inhibitors, and vaccine therapy.     

Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing

Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients’ plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.     

Next-generation sequencing of FLT3 internal tandem duplications for minimal residual disease monitoring in acute myeloid leukemia

Minimal Residual Disease (MRD) detection can be used for early intervention in relapse, risk stratification, and treatment guidance. FLT3 ITD is the most common mutation found in AML patients with normal karyotype. We evaluated the feasibility of NGS with high coverage (up to 2.4.10⁶ PE fragments) for MRD monitoring on FLT3 ITD. We sequenced 37 adult patients at diagnosis and various times of their disease (64 samples) and compared the results with FLT3 ITD ratios measured by fragment analysis. We found that NGS could detect variable insertion sites and lengths in a single test for several patients. We also showed mutational shifts between diagnosis and relapse, with the outgrowth of a clone at relapse different from that dominant at diagnosis. Since NGS is scalable, we were able to adapt sensitivity by increasing the number of reads obtained for follow-up samples, compared to diagnosis samples. This technique could be applied to detect biological relapse before its clinical consequences and to better tailor treatments through the use of FLT3 inhibitors. Larger cohorts should be assessed in order to validate this approach.     

Insights into minimal residual disease in cancer patients: Implications for anti-cancer therapies

Tumor cell dissemination appears even in patients with small solid tumors, and bone marrow (BM) is a common homing organ for disseminated tumor cells (DTC) derived from various types of primary epithelial tumors. Tumor cells are frequently detected in the BM of cancer patients without any clinical or even histopathological signs of overt metastases. It is crucial, however, to improve and standardize methods for the detection of DTC.The characterization of DTC has shed new light on the process underlying early tumor cell dissemination and metastatic progression in cancer patients. Characterization of DTC should help to identify novel targets for biological therapies aimed at preventing metastatic relapse and to monitor the efficacy of these therapies. Evidence has emerged that the detection of DTC and circulating tumor cells (CTC) in blood may provide important prognostic information and, in addition, might help to monitor the efficacy of therapy.In this article, we summarize the clinical background and the technical aspects of current methods used for the detection and characterization of DTC in BM and CTC in blood, with a special focus on breast cancer.     

Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples

BackgroundNext-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts.Patients and methodsWe have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies.ResultsUsing matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data.ConclusionWe strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation.     

Targeted next-generation sequencing reveals molecular heterogeneity in non-chronic lymphocytic leukemia clonal B-cell lymphocytosis

Non-chronic lymphocytic leukemia (non-CLL) clonal B-cell lymphocytosis (CBL) encompasses a heterogeneous group of hematologic disorders that are still poorly understood. To shed light on their biological aspects, we retrospectively analyzed a highly selected series of 28 patients, who had a clonal B-cell population in the peripheral blood and in the bone marrow, without evidence of lymphoma. Extended targeted next-generation sequencing revealed wide molecular heterogeneity with MYD88 (14%), PDE4DIP (14%), BIRC3 (11%), CCND3 (11%), NOTCH1 (11%), and TNFAIP3 (11%) as the most mutated genes. Mutations of MYD88 were “nonclassic” in most cases. Although some genetic lesions were overlapping with indolent lymphomas, mainly splenic B-cell lymphomas of marginal zone origin and splenic diffuse red pulp small B-cell lymphoma, the genetic profile of our non-CLL CBL series seemed to suggest that various pathways could be involved in the pathogenesis of these disorders, not mirroring any specific lymphoma entity. These data better enlighten the molecular characteristics of non-CLL CBL; however, more efforts are needed in order to improve the diagnostic process, prognostication, and clinical management.     

Molecular diagnosis of minimal residual disease in head and neck cancer patients

Aim

Locoregional recurrences and distant metastases in adequately treated head and neck squamous cell carcinoma (HNSCC) patients have a dismal effect on survival. Tumor cells that escape histopathological detection might be the prime cause of this effect. We evaluated whether minimal residual cancer (MRC) in deep surgical margins and disseminated tumor cells (DTCs) in bone marrow aspirates are associated with clinicohistopathological parameters and outcome.

Methods

Submucosal samples of deep resection margins of 105 HNSCC patients with histopathologically tumor-free surgical margins were analysed for the presence of MRC using hLy-6D qRT-PCR. Bone-marrow aspirates of 76 of these patients were analysed for DTCs by immunocytochemical staining. Presence of molecular-positive deep surgical margins, presence of DTC in bone marrow aspirates, and clinicohistopathological parameters were tested for associations with survival parameters by univariate and multivariate analyses.

Results

In addition to lymph node stage, it appeared that vasoinvasive growth and particularly infiltrative growth pattern are significant predictors for locoregional recurrence (p?=?0.041 and p?=?0.006, respectively) and disease?Cfree survival (p?=?0.014 and p?=?0.008, respectively). Remarkably, neither the presence of molecular-positive deep surgical margins nor that of DTC in bone marrow aspirates were significantly related to outcome.

Conclusions

The presence of vasoinvasive and infiltrative growth in HNSCC tumor specimens are significant risk-factors for locoregional recurrence and disease-free survival. At present there seems no role for molecular analysis of deep surgical margins and bone marrow aspirates in predicting outcome with the methods used.     

Targeted agents for adjuvant therapy of colon cancer

Among patients with colorectal cancer (CRC) diagnosed in the United States, 37.2% are diagnosed with stage III and 27.9% with stage II disease. In locoregionally advanced CRC, surgery is the primary treatment modality and has a curative intent. The survival depends on the pathologic stage and varies from 30%-60% for stage III to 60%-80% for stage II. However, as much as 40%-50% of patients will relapse and require additional treatment of the disease. Clinical failure after resection of CRC is predominantly secondary to the clinical progression of previously undetected distant metastatic disease. Until very recently, the absolute benefit for survival obtained with adjuvant therapy compared with control was about 6%. Introduction of oxaliplatin in the adjuvant setting has shown a reduction of 23% in the risk of relapse when compared with 5-fluorouracil alone (MOSAIC). Recent phase III studies have shown that targeted agents improved survival in patients with advanced-stage CRC. Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor, is the first antiangiogenic drug to show improved efficacy when used in combination with irinotecan and oxaliplatin for first- and second-line treatment of CRC. Cetuximab, another monoclonal antibody targeting epidermal growth factor receptor, has shown efficacy in third-line therapy and promising results in first-line phase II studies. There is great interest in whether the biologic agents bevacizumab and cetuximab can improve survival in the adjuvant-therapy setting. This article reviews the adjuvant therapy for colon cancer and discusses the potential role and current trials involving the targeted agents.     

二代测序在家族遗传性高危胃肠肿瘤筛查中的应用

  目的  探索二代测序技术对家族遗传性高危胃肠肿瘤患者进行遗传筛查的意义及高危因素在筛选患者中的价值。  方法  选取2016年3月至2016年4月收治于北京大学肿瘤医院的322例结直肠癌及胃癌患者,筛选出25例遗传性胃肠肿瘤高危患者,运用二代测序技术对患者的外周血白细胞DNA进行42个遗传性肿瘤综合征相关基因的胚系检测。  结果  24%（6/25）患者检测出遗传性肿瘤相关基因的病理性胚系突变,其中50%（3/6）患者肿瘤组织的免疫组织化学检测表现为错配修复蛋白表达缺失,83%（5/6）患者发病年龄≤50岁且具有恶性肿瘤家族史。发生胚系突变的6例遗传性肿瘤相关基因分别为MYH基因错义突变1例,APC基因缺失突变1例和遗传性非息肉病性结直肠癌（hereditary nonpolyposis colorectal cancer,HNPCC）相关基因的突变4例（包括MLH1、MLH3、TGFBR2的错义突变和MSH6的无义突变各1例）,且提供了MLH3的胚系致病突变的家系验证。  结论  通过二代测序技术对本研究入组的25例患者进行家族遗传性肿瘤综合征的筛查,检测出遗传性肿瘤相关基因的胚系致病突变6例,提示运用二代测序技术对家族遗传性高危消化道肿瘤患者进行遗传筛查具有提高检测阳性率的临床应用价值。      

Examination of the differences between sulforaphane and sulforaphene in colon cancer: A study based on next-generation sequencing

Sulforaphane and sulforaphene are isothiocyanate compounds derived from cruciferous vegetables that have demonstrated antiproliferative properties against colon cancer. However, the underlying mechanism of action of these two compounds has yet to be elucidated. The aim of the present study was to examine the effects of sulforaphane and sulforaphene on colon cancer using next-generation sequencing (NGS). The SW480 colon cancer cell line was cultured with 25 µmol/l sulforaphane or sulforaphene. Total RNA was extracted from the cells following 48 h of incubation with these compounds, and NGS was performed. Pearson''s correlation and principal component analyses were performed on the NGS data in order to determine sample homogeneity followed by hierarchical clustering, chromosomal location, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A total of 873 probes in the sulforaphene group were differentially expressed compared with the control group. Similarly, 959 probes in the sulforaphane group were differentially expressed compared with the control group. The differentially expressed genes were dispersed on the chromosomes, across 22 pairs of autosomes, as well as the X and Y chromosomes. GO and KEGG analyses demonstrated that both drugs affected the ‘p53 signaling pathway’, ‘MAPK signaling pathway’, ‘FOXO signaling pathway’ and ‘estrogen signaling pathway’, while ‘Wnt signaling pathway’ was enriched in the sulforaphane group, and ‘ubiquitin mediated proteolysis’ and ‘estrogen signaling pathway’ in the sulforaphene group. Thus, sulforaphane and sulforaphene exhibited similar biological activities on colon cancer cells. Sulforaphane and sulforaphene may be associated with Wnt and estrogen signaling, respectively.